Carbonyl reductase and application thereof

Abstract

The invention provides carbonyl reductase and application thereof. The carbonyl reductase is specifically provided, and compared with SEQ ID NO: 1, the amino acid sequence of the carbonyl reductase contains mutation of the following sites: G93A, S152I, Y188A and H202I. The carbonyl reductase provided by the invention is good in stereoselectivity, high in conversion rate and good in application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a carbonyl reductase and application thereof. BACKGROUND

[0002] Iptacopan is the first targeted inhibitor of complement factor B developed by Novartis, Switzerland. It was developed in 2014 and granted breakthrough drug qualification by the US Food and Drug Administration (FDA) for the treatment of PNH in December 2020. On December 5, 2023, the FDA approved Iptacopan capsules for the treatment of adult PNH patients, becoming the first FDA-approved oral drug for the treatment of PNH.

[0003] (2S,4S)-benzyl-4-ethoxy-2-(4-(methyl ester(methoxycarbonyl)) phenyl) piperidine-1-carboxylate is a key intermediate of iptacopan, which has two chiral centers.

[0004] In the preparation of the key intermediate, the patent (CN112513025A) uses 4-iodobenzoic acid methyl ester, 4-methoxypyridine, and benzyl chloroformate to couple and reduce the double bond to obtain benzyl-2-(4-(methyl ester(methoxycarbonyl)) phenyl)-4-carbonyl piperidine-1-carboxylate, which has one chiral center, resulting in the generation of two isomers, which need to be chiral split to obtain a single configuration, resulting in a large loss of intermediates. Currently, there is no separate synthesis route reported for isomer 2.

[0005]

[0006] The patent (CN117500499A) introduces another chiral center after reducing the ketone carbonyl, resulting in the generation of four isomers, which need to be chiral split to obtain a single configuration of the product. Currently, there is no separate synthesis route reported for isomers 4, 5, and 6.

[0007]

[0008] In summary, a high-selectivity synthesis method for the above isomers is provided, which is particularly important for industrial production and drug research. SUMMARY

[0009] The technical problem to be solved by the present application is to overcome the lack of preparation methods for iptacopan intermediate isomers. To this end, the present application provides a carbonyl reductase and application thereof. The ketoreductase provided by the present application can induce high selectivity of chirality, prepare chiral isomers with high chirality purity, and has good application prospect.

[0010] The present application solves the above technical problems through the following scheme.

[0011] The present application provides a carbonyl reductase, wherein the amino acid sequence of the carbonyl reductase comprises a mutation selected from G93A, S152I, Y188A and H202I, compared with SEQ ID NO: 1.

[0012] In an embodiment, the amino acid sequence of the carbonyl reductase has G93A, S152I, Y188A and H202I mutations, compared with SEQ ID NO: 1.

[0013] In an embodiment, the carbonyl reductase has the amino acid sequence as shown in SEQ ID NO: 3.

[0014] The present application provides a polynucleotide encoding the carbonyl reductase as described in the first aspect of the present application.

[0015] In an embodiment, the polynucleotide has the sequence as shown in SEQ ID NO: 4.

[0016] The present application provides a recombinant expression vector comprising the polynucleotide as described in the second aspect of the present application.

[0017] In an embodiment, the recombinant expression vector is a plasmid. Preferably, the backbone of the plasmid is pET28a(+).

[0018] The present application provides a transformant comprising the polynucleotide as described in the second aspect of the present application or the recombinant expression vector as described in the third aspect of the present application.

[0019] In an embodiment, the transformant is a cell, preferably a prokaryotic cell. The prokaryotic cell can be an E. coli cell. For example, the transformant is an E. coli cell, and the E. coli is preferably E. coli BL21(DE3).

[0020] The present application provides a method for preparing the carbonyl reductase as described in the first aspect of the present application, comprising culturing the transformant as described in the fourth aspect of the present application under conditions suitable for expression.

[0021] In an embodiment, the culture medium comprises the following components: 5-17 g / L of yeast extract, 10-22 g / L of tryptone, 3-13 g / L of NaCl and water. Preferably, the culture medium comprises the following components: 5-17 g / L of yeast extract, 10-22 g / L of tryptone, 3-13 g / L of NaCl, 5-15 g / L of glycerol, 0.5-4 g / L of K2HPO4·3H2O, 0.1-2 g / L of MgSO4·7H2O and water. Further preferably, the water is deionized water.

[0022] In an embodiment, the culture medium is LB liquid medium and / or fermentation medium, for example, the fermentation medium has a formulation of 12 g / L yeast extract, 12 g / L tryptone, 3 g / L NaCl, 10 g / L glycerol, 2 g / L K2HPO4·3H2O, 0.5 g / L MgSO4·7H2O and deionized water, and for example, the LB liquid medium has a formulation of 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl and deionized water.

[0023] In an embodiment, the culture medium further comprises an antibiotic, for example, kanamycin. The antibiotic can be present in an amount of 50 μg / mL.

[0024] In an embodiment, the culture is performed at a temperature of 25-37 °C, for example, 37 °C.

[0025] In an embodiment, the culture is performed at a pH of 7.

[0026] The sixth aspect of the present application provides a use of a carbonyl reductase in the preparation of a compound as shown in Formula I,

[0027]

[0028] the carbon atom marked with “*” represents S configuration, R configuration or a mixture thereof when it is a chiral carbon atom;

[0029] R 1 is H, C6-C 10 aryl or C6-C 1-1 substituted C6-C 10 aryl;

[0030] R 1-1 is cyano or wherein R 1-2 is C1-C6 alkyl;

[0031] R 2 is H or an amino protecting group;

[0032] The carbonyl reductase is (1) the above-mentioned carbonyl reductase,

[0033] and / or (2) a carbonyl reductase having an amino acid sequence as shown in SEQ ID NO: 1.

[0034] In an embodiment, R 1 , the C6-C 10 aryl and C6-C 1-1 substituted C6-C 10 aryl, the C6-C 10 aryl is each independently phenyl or naphthyl, for example, phenyl.

[0035] In one aspect, R 1-2 In one aspect, the C1-C6 alkyl is methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl or t-butyl, for example methyl.

[0036] In one aspect, the amino protecting group is a conventional amino protecting group in the art, for example Boc or Cbz.

[0037] In one aspect, the carbon atom marked with "*" is an S-configuration or R-configuration carbon atom.

[0038] In one aspect, R 1 is C6-C10 aryl substituted with one R 1-1 In one aspect, the C6-C10 aryl is phenyl. 10 In one aspect, the C6-C10 aryl is phenyl. In one aspect, the C6-C10 aryl is phenyl.

[0039] In one aspect, R 1-1 is C6-C10 aryl substituted with one R

[0040] In one aspect, R 2 is an amino protecting group, preferably Cbz

[0041] In one aspect, the compound of formula I is

[0042] In a seventh aspect, the present application provides a method for preparing a compound of formula I, comprising the following steps: subjecting a compound of formula II to a reduction reaction as shown in the following scheme in the presence of coenzyme and carbonyl reductase in a liquid reaction system to obtain a compound of formula I;

[0043]

[0044] wherein the carbonyl reductase, the carbon atom marked with "*", R 1 and R 2 are each independently as described in the sixth aspect of the present application.

[0045] In one aspect, the liquid reaction system comprises a buffer. The buffer is preferably a phosphate buffer, for example 0.1 M phosphate buffer.

[0046] In one aspect, the liquid reaction system further comprises a cosolvent. The cosolvent can be a conventional cosolvent in the art, and is preferably selected from one or more of dimethyl sulfoxide, isopropanol and toluene, for example dimethyl sulfoxide.

[0047] In one aspect, the liquid reaction system further comprises a hydrogen donor, for example glucose.

[0048] In one embodiment, the coenzyme is a conventional coenzyme in the art, and the coenzyme can be a reduced coenzyme, such as NADH and / or NADPH, preferably, the coenzyme is an oxidized coenzyme, such as NAD+ and / or NADP+, and a dehydrogenase, such as glucose dehydrogenase. The reduced coenzyme can be obtained by a coenzyme regeneration step, in which NADP+ is reduced to NADPH in the presence of glucose dehydrogenase and a hydrogen donor, such as glucose. + and / or NAD + .

[0049] In one embodiment, the glucose dehydrogenase is derived from a glucose dehydrogenase of Bacillus megaterium, and the glucose dehydrogenase preferably has an amino acid sequence as shown in SEQ ID NO: 5, and a polynucleotide encoding the glucose dehydrogenase preferably has a sequence as shown in SEQ ID NO: 6.

[0050] In one embodiment, the compound of Formula II is

[0051] In one embodiment, the amount of the buffer can be a conventional amount of the buffer in the art, and preferably, the volume to mass ratio of the buffer to the compound of Formula II is 5-100 mL / g, such as 37.5 mL / g.

[0052] In one embodiment, the amount of the co-solvent can be a conventional amount of the co-solvent in the art, and preferably, the volume to mass ratio of the co-solvent to the compound of Formula II is 1-20 mL / g, such as 12.5 mL / g.

[0053] In one embodiment, the coenzyme comprises NAD+ and / or NADP+, and the amount of the NAD+ and / or NADP+ is a conventional amount of the coenzyme in the art, and preferably, the mass ratio of the NADP+ to the compound of Formula II is (0.001-0.1): 1, preferably (0.01-0.05): 1, such as 0.02: 1.

[0054] In one embodiment, the coenzyme comprises glucose dehydrogenase, and the amount of the glucose dehydrogenase is conventional in the art, and the mass ratio of the glucose dehydrogenase to the compound of Formula II is preferably (1-5): 1, preferably (2-3): 1, such as 2.5: 1, in terms of the added mass of the dehydrogenase calculated from the mass of the wet bacteria producing the dehydrogenase.

[0055] In one embodiment, the molar ratio of the hydrogen donor to the compound of Formula II is (1-5): 1, preferably 2: 1.

[0056] In one embodiment, the carbonyl reductase is added to the reduction reaction in a form conventional in the art, preferably as a free enzyme, an immobilized enzyme, a bacterial powder or a bacterial cell, such as a bacterial cell, preferably the transformant of the fourth aspect of the application. Further preferably, the mass ratio of the bacterial cell to the compound of formula II is (1-10): 1, preferably (4-6): 1, such as 5: 1.

[0057] In one embodiment, the reduction reaction is carried out at a temperature conventional in the art, preferably at a temperature of 10°C to 50°C, such as 25°C or 30°C.

[0058] In one embodiment, the progress of the reduction reaction is monitored by a method conventional in the art, such as TLC, for example, until the compound of formula II is completely reacted or no longer reacts. For example, the reaction time is 1-48 hours, preferably 24 hours.

[0059] In one embodiment, the reduction reaction further comprises a work-up after the reduction reaction.

[0060] The work-up can be a work-up conventional in the art, preferably, the work-up comprises the following steps: adding a halogenated alkane solvent to the liquid reaction system, extracting, and isolating the compound of formula I. The halogenated alkane solvent can be dichloromethane. The isolation can comprise concentration (for example, concentration under reduced pressure), extraction with an ester solvent (for example, ethyl acetate), washing (for example, washing with saturated brine), drying (for example, drying over anhydrous sodium sulfate), filtration, and concentration steps to obtain the compound of formula I.

[0061] In one embodiment, the reduction reaction comprises the following steps: mixing the compound of formula II, the cosolvent (for example, dimethyl sulfoxide), the buffer solvent (for example, phosphate buffer), NADP+, glucose, the glucose dehydrogenase (for example, a cell producing glucose dehydrogenase), and the carbonyl reductase (for example, the transformant) mixture at a temperature of 10°C to 50°C to carry out the reduction reaction, and after the reaction, adding a halogenated alkane solvent, extracting, and isolating the compound of formula I.

[0062] As understood by those skilled in the art, the use of means that the corresponding group is connected to other fragments, groups in the compound via this site.

[0063] The term "aryl" refers to a cyclic group consisting only of carbon atoms, having the specified number of carbon atoms (e.g., C6-10, C6-14), which is monocyclic or polycyclic, and at least one ring of which is aromatic (complies with Huckel's rule). The aryl group is attached to the rest of the molecule by a ring having aromaticity or a ring not having aromaticity. The aryl group includes, but is not limited to, phenyl, naphthyl, and the like.

[0064] The term "alkyl" refers to a straight-chain or branched alkyl group having the specified number of carbon atoms (e.g., C1-C6). The alkyl group includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0065] The above-mentioned preferred conditions can be combined arbitrarily without departing from the common general knowledge in the art, thereby obtaining preferred embodiments of the present application.

[0066] The reagents and raw materials used in the present application are commercially available.

[0067] The positive progress effect of the present application is that the reductase of the present application has high conversion rate, high chiral purity, simple post-treatment, low cost, green environmental protection, and is suitable for industrial production when used for preparing 4-hydroxypiperidine compounds. DETAILED DESCRIPTION

[0068] The present application is further illustrated by the following examples without limiting the present application to the examples. The experimental methods in the following examples are not specified, and are selected according to conventional methods and conditions or according to the product instructions.

[0069] Example 1: Construction of genetically engineered bacteria expressing recombinant carbonyl reductase and glucose dehydrogenase

[0070] The wild-type carbonyl reductase EsADH target gene (nucleotide sequence is SEQ ID NO: 2, and amino acid sequence is SEQ ID NO: 1), the mutant carbonyl reductase EsADH-E5 target gene (nucleotide sequence is SEQ ID NO: 4, and amino acid sequence is SEQ ID NO: 3), and the glucose dehydrogenase GDH target gene (nucleotide sequence is SEQ ID NO: 6, and amino acid sequence is SEQ ID NO: 5) derived from Bacillus megaterium were subjected to whole gene synthesis, and were respectively cloned into the pET28a(+) vector, and were transformed into the competent cells of Escherichia coli BL21(DE3), and single bacteria were picked and cultured in LB to obtain the EsADH bacterial body, the genetically engineered bacteria pET28a(+)-EsADH-E5 capable of inducing expression of recombinant carbonyl reductase, and the genetically engineered bacteria pET28a(+)-GDH expressing recombinant glucose dehydrogenase GDH.

[0071] Example 2: Preparation of recombinant carbonyl reductase, glucose dehydrogenase

[0072] The genetically engineered bacteria saved in the previous step were inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C, 220 rpm for 14 h to obtain seed culture. The seed culture was inoculated into LB liquid medium containing 50 μg / mL kanamycin resistance at a ratio of 0.5%, and then cultured at 37°C, 220 rpm until the OD 600 The final concentration of isopropyl thiogalactoside (IPTG) was 0.5 mM, and the temperature was reduced to 25°C to induce protein expression. The culture was continued for 20 h, and then the tank was removed and centrifuged to obtain the bacterial cells, which were prepared for biotransformation.

[0073] LB liquid medium (g / L): tryptone 10.0 g, yeast extract 5.0 g, NaCl 10.0 g, deionized water 1 L, pH 7.0.

[0074] Fermentation medium (g / L): yeast extract 12.0 g, tryptone 12.0 g, NaCl 3.0 g, glycerol 10.0 g, K2HPO4·3H2O 2.0 g, MgSO4·7H2O 0.5 g, deionized water 1 L, pH 7.0.

[0075] Example 3: Preparation of benzyl (2S,4R)-4-hydroxy-2-(4-(methyl ester <methoxycarbonyl>) phenyl) piperidine-1-carboxylate and benzyl (2R,4R)-4-hydroxy-2-(4-(methyl ester <methoxycarbonyl>) phenyl) piperidine-1-carboxylate by different carbonyl reductases biocatalysis

[0076] Each benzyl (S)-2-(4-(methyl ester <methoxycarbonyl>) phenyl)-4-oxopiperidine-1-carboxylate (10 mg, 0.027 mmol, 1 eq.) was dissolved in DMSO (0.125 mL), 0.1 M phosphate buffer (0.375 mL) was added, NADP+ (0.1 mg) was added, and glucose (10.8 mg, 0.054 mmol, 2 eq.) was added. One part was EsADH bacterial cells (50 mg), and the other part was EGDH bacterial cells (25 mg). The reaction was carried out at 25°C on a constant temperature shaker at 220 rpm for 2 h, and the conversion rate was monitored by liquid phase. It was observed that the conversion rate of the raw material added with wild-type EsADH bacterial cells was 80%, and the ee value was 99.99%; the conversion rate of the raw material added with mutant EsADH-E5 bacterial cells was 100%, and the ee value was 99.99%.

[0077] The reaction results are shown in the following table:

[0078] Enzyme Conversion (%) R configuration ee value (%) EsADH 80 99.99 EsADH-E5 100 99.99

[0079] Example 4: Synthesis of benzyl (R)-2-(4-(methoxycarbonyl)phenyl)-4- oxypiperidine-1-carboxylate

[0080]

[0081] Benzyl 4-oxo-3,4-dihydropyridine-1(2H)-carboxylate (10.0 g, 43.4 mmol, 1.0 eq), 4-methoxycarbonylphenylboronic acid pinacol ester (45.4 g, 173.1 mmol, 4.0 eq), (R)-2,2'-bis[di(3,5-dimethylphenylphosphine)]-1,1'-binaphthalene ((R)xylBINAP) (2.4 g, 3.3 mmol, 0.075 eq), acetylacetonato bis(ethylene) rhodium (I) (Rh(Acac)(C2H4)2) (336.0 mg, 1.3 mmol, 0.03 eq), cesium carbonate (42.3 g, 129.7 mmol, 3.0 eq) were placed in a 1 L three-necked flask, 1,4-dioxane (200 mL) and water (40 mL) were added, nitrogen was replaced for three times, then the reaction was heated to reflux at 92 °C, the reaction solution was dark wine red. TLC monitoring (PE:EA = 3:1), 48 h reaction was basically completed, after the reaction was completed, it was cooled to room temperature, concentrated under reduced pressure, then dissolved in ethyl acetate, filtered, the filter cake was washed to white, the filtrate was concentrated under reduced pressure and recrystallized with n-heptane: methyl tert-butyl ether = 3:1 to obtain 8.1 g of white solid. HRMS (ESI) m / z: calculated C 21 H 21 NO5 + [M+H] + 368.14, [M+Na] + 390.14, found 368.14823, 390.12984. 1 H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 8.4 Hz, 2H), 7.34 (dd, J = 21.4, 15.1 Hz, 7H), 5.82 (s, 1H), 5.28-5.14 (m, 2H), 4.30 (s, 1H), 3.91 (s, 3H), 3.23 (dd, J = 14.6, 7.3 Hz, 1H), 2.94 (ddd, J = 22.3, 15.5, 5.0 Hz, 2H), 2.62-2.50 (m, 1H), 2.38 (d, J = 16.2 Hz, 1H). 13C NMR (101 MHz, CDC13) δ 206.66 (s), 166.56 (s), 155.44 (s), 136.04 (s), 130.17 (s), 129.69 (s), 128.62 (s), 128.36 (s), 128.07 (s), 126.62 (s), 67.99 (s), 54.65 (s), 52.21 (s), 40.45 (s), 39.24 (s).

[0082] Benzyl (S)-2-(4-(carbomethoxy)phenyl)-4-oxopiperidine-l-carboxylate can be prepared according to the published method A Practical Method for Synthesizing Iptacopan.

[0083] Example 5: Synthesis of Benzyl (2S,4R)-4-hydroxy-2-(4- (carbomethoxy)phenyl)piperidine-l-carboxylate

[0084]

[0085] Benzyl (S)-2-(4-(carbomethoxy)phenyl)-4-oxopiperidine-l-carboxylate (10.0 g, 27.2 mmol, 1 eq) was dissolved in DMSO (125.0 mL), 0.1 M phosphate buffer (375.0 mL) was added, NADP+ (0.2 g) was added, glucose (2 eq.) was added, EsADH-E5 bacteria (50.0 g) and EGDH bacteria (25.0 g) were added, and the reaction was carried out at 25°C under constant shaking at 220 rpm. TLC monitoring (PE:EA = 1:1) showed that the reaction was essentially complete after 24 h. The reaction was quenched by adding dichloromethane (500 mL) and the product was extracted, centrifuged, and the organic phase was concentrated under reduced pressure, replaced with ethyl acetate solvent, and washed with saturated brine three times. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give 6.6 g of white solid (yield 65.6%, purity 99%, ee value 99.99%). HRMS (ESI) m / z: calculated C 21 H 23 NO5 + [M+H] + 370.16, [M+Na] + 392.16, found 370.16378, 392.14541. 1H NMR (400MHz, CDCl3) δ7.99(d,J=8.4Hz,2H),7.37-7.28(m,5H),7.27-7.22(m,2H),5.41(dd,J=6.4,3.2Hz,1H),5.14(s,2H),4.23-4.08(m,2H),3.9 1(s,3H),3.41(td,J=13.2,3.4Hz,1H),2.44(dt,J=14.5,3.0Hz,1H),2.20 (ddd,J=14.6,6.8,3.2Hz,1H),1.95-1.83(m,1H),1.68(d,J=12.6Hz,1H). 13 C NMR(101MHz, CDCl3)δ166.84(s),155.92(s),144.81(s),136.42(s),130.08(s),128.83(s),128.56(s),12 8.17(s),127.87(s),126.19(s),67.65(s),64.71(s),53.81(s),52.19(s),39.32(s),37.02(s),34.77(s).

[0086] Example 6: Synthesis of benzyl(2R,4R)-4-hydroxy-2-(4-(methyl ester <methoxycarbonyl>)phenyl)piperidine-1-carboxylic acid ester

[0087]

[0088] Dissolve (R)-2-(4-(methyl ester <methoxycarbonyl>)phenyl)-4-carbonylpiperidin-1-carboxylic acid ester (10.0 g, 27.2 mmol, 1.0 eq) in DMSO (125.0 mL), add 0.1 M phosphate buffer (375 mL), and add NADP. + 0.2 g of glucose (2 eq.) was added to EsADH-E5 cells (50.0 g) and GDH cells (25.0 g). The mixture was reacted in a shaker at 220 rpm at 25 °C, monitored by TLC (PE:EA = 1:1). The reaction was essentially complete after 24 h. Dichloromethane (500.0 mL) was added to quench the reaction and extract the product. The mixture was centrifuged, and the organic phase was concentrated under reduced pressure. The solution was replaced with ethyl acetate and washed three times with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give 6.6 g of a white solid (yield 65.6%, purity 99%, ee value 99.99%). HRMS (ESI) m / z: calculated value C 21 H 23 NO5 + [M+H] +370.16, [M+Na] + 392.16, found 370.16384, 392.14684. 1 H NMR (400 MHz, CDC13) δ 7.98 (d, J = 8.4 Hz, 2H), 7.29 (dd, J = 13.2, 8.3 Hz, 7H), 5.67 (d, J = 3.1 Hz, 1H), 5.20 (d, J = 12.1 Hz, 2H), 4.29 - 4.06 (m, 1H), 3.90 (s, 3H), 3.78 - 3.66 (m, 1H), 2.82 (td, J = 13.6, 2.8 Hz, 1H), 2.68 - 2.58 (m, 1H), 1.84 (ddd, J = 13.3, 11.6, 5.8 Hz, 2H), 1.49 (ddd, J = 23.8, 12.7, 4.8 Hz, 1H), 1.31 - 1.22 (m, 1H). 13 C NMR (101 MHz, CDC13) δ 166.84 (s), 155.92 (s), 144.81 (s), 136.42 (s), 130.08 (s), 128.83 (s), 128.56 (s), 128.17 (s), 127.87 (s), 126.19 (s), 67.65 (s), 64.71 (s), 53.81 (s), 52.19 (s), 39.32 (s), 37.02 (s), 34.77 (s).

[0089] SEQ ID NO: 1:

[0090] MSILKDKVAIVTGAGSGIGKAVAELYAKEGAKVVVSDINEEGGKEVVEIIKKNGGEAFFFKADTALAEDNEALVNKAVEVYGKLDIACNNAGIGGPAALTEDYPLDGWKKVIDINFNGVFYGCKYQLQAMEKNGGGAIVNMASIHGEVAAPMSSAYTSAKHGVVGLTKNIGAEYGPKNIRCNAVGPGYIMTPLLSNNLSADHLEVLVTKHPMGRLGQPEEVAELVLFLSSDKASFMTGAYYLVDGGYTAV

[0091] SEQ ID NO: 2:

[0092] ATGTCAATTCTAAAGGATAAAGTAGCTATAGTGACGGGTGCTGGCAGCGGTATTGGCAAGGCGGTAGCGGAACTGTACGCAAAGGAGGGCGCGAAGGTCGTGGTGTCGGACATCAACGAAGAGGGTGGCAAAGAGGTCGTGGAAATTATCAAAAAGAACGGTGGAGAGGCGTTTTTCTTCAAAGCTGACACCGCGCTGGCGGAGGATAATGAAGCACTCGTCAATAAGGCCGTCGAGGTTTATGGTAAGCTGGATATTGCGTGTAACAATGCGGGCATCGGTGGTCCGGCAGCGCTTACCGAAGACTACCCGTTGGATGGTTGGAAAAAGGTGATTGATATCAATTTCAACGGCGTTTTTTATGGCTGCAAATACCAGCTGCAGGCAATGGAAAAAAATGGTGGCGGTGCGATTGTTAACATGGCATCCATCCACGGCGAGGTGGCAGCTCCGATGAGCAGCGCGTATACGAGCGCTAAGCACGGCGTTGTTGGTCTGACCAAAAACATCGGCGCTGAGTATGGTCCGAAAAACATCCGCTGCAACGCCGTGGGCCCAGGTTACATTATGACCCCGTTGCTGTCTAATAACCTGTCTGCCGACCATCTGGAAGTTTTGGTTACCAAGCATCCGATGGGTCGTTTAGGTCAACCGGAAGAGGTTGCGGAACTGGTGCTGTTCCTGTCCAGCGACAAAGCCAGCTTTATGACCGGTGCGTACTACCTGGTTGATGGCGGCTATACTGCGGTGTAA

[0093] SEQ ID NO: 3

[0094] MSILKDKVAIVTGAGSGIGKAVAELYAKEGAKVVVSDINEEGGKEVVEIIKKNGGEAFFFKADTALAEDNEALVNKAVEVYGKLDIACNNAGIAGPAALTEDYPLDGWKKVIDINFNGVFYGCKYQLQAMEKNGGGAIVNMASIHGEVAAPMISAYTSAKHGVVGLTKNIGAEYGPKNIRCNAVGPGAIMTPLLSNNLSADILEVLVTKHPMGRLGQPEEVAELVLFLSSDKASFMTGAYYLVDGGYTAV*

[0095] SEQ ID NO: 4

[0096] ATGTCAATTCTAAAGGATAAAGTAGCTATAGTGACGGGTGCTGGCAGCGGTATTGGCAAGGCGGTAGCGGAACTGTACGCAAAGGAGGGCGCGAAGGTCGTGGTGTCGGACATCAACGAAGAGGGTGGCAAAGAGGTCGTGGAAATTATCAAAAAGAACGGTGGAGAGGCGTTTTTCTTCAAAGCTGACACCGCGCTGGCGGAGGATAATGAAGCACTCGTCAATAAGGCCGTCGAGGTTTATGGTAAGCTGGATATTGCGTGTAACAATGCGGGCATCgcgGGTCCGGCAGCGCTTACCGAAGACTACCCGTTGGATGGTTGGAAAAAGGTGATTGATATCAATTTCAACGGCGTTTTTTATGGCTGCAAATACCAGCTGCAGGCAATGGAAAAAAATGGTGGCGGTGCGATTGTTAACATGGCATCCATCCACGGCGAGGTGGCAGCTCCGATGatcAGCGCGTATACGAGCGCTAAGCACGGCGTTGTTGGTCTGACCAAAAACATCGGCGCTGAGTATGGTCCGAAAAACATCCGCTGCAACGCCGTGGGCCCAGGTgctATTATGACCCCGTTGCTGTCTAATAACCTGTCTGCCGACattCTGGAAGTTTTGGTTACCAAGCATCCGATGGGTCGTTTAGGTCAACCGGAAGAGGTTGCGGAACTGGTGCTGTTCCTGTCCAGCGACAAAGCCAGCTTTATGACCGGTGCGTACTACCTGGTTGATGGCGGCTATACTGCGGTGTAA

[0097] SEQ ID NO: 5

[0098] MYPDLKGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDVINLVQSAIKEFGKLDIMINNAGLENPVSSHEMSLSDWNKVIDTNLTGAFLGSREAIKYFVENDVKGTVINMSSVHEKIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTQYPSFQAGRG

[0099] SEQ ID NO: 6

[0100] atgtatccggatttaaaaggaaaagtagttgtcataacaggttcatctacaggtttgggaaaatcaatggcgattcgttttgcgacagaaaaagccaaagtagttgtgaattatcgttcgaaagaagacgaagctaacagcgtcttagaagaaattaaaaaagttggcggagaggcaattgccgtcaaaggtgatgtaacagttgagtctgacgttatcaatttagttcaatctgctattaaagaatttggaaagctagacattatgattaacaacgcagggttagaaaatccggtttcatctcatgaaatgtctttaagtgactggaataaagtcattgatacgaacttaacgggagcattctt

[0101] aggcagccgtgaagcgattaaatattttgtagaaaatgatgttaagggaacagttattaacatgtcgagtgttcacgagaaaattccttggcc

[0102] attatttgttcattacgcagcaagtaaaggcggtatgaagctcatgactgaaacacttgcattagaatacgctccaaaaggcattcgtgtaaa

[0103] taacattggaccgggagcgattaatacaccgattaacgctgagaaatttgctgatcctgagcagcgtgcagatgtagaaagcatgattcca

[0104] atgggatacatcggagagccggaagaaattgcagcggttgctgcatggctagcttcttcagaggcaagttatgtaacagggattacgctc

[0105] tttgctgacggcggtatgacacagtacccatcattccaggcaggccgcggttaa。

Claims

1. A carbonyl reductase having an amino acid sequence comprising, compared to SEQ ID NO:1, mutations selected from the following sites: G93A, S152I, Y188A and H202I.

2. The carbonyl reductase as described in claim 1, characterized in that, The amino acid sequence of the carbonyl reductase, compared to SEQ ID NO:1, has mutations in G93A, S152I, Y188A, and H202I; Preferably, the carbonyl reductase has the amino acid sequence shown in SEQ ID NO:

3.

3. A polynucleotide, characterized in that, The polynucleotide encodes the carbonyl reductase as described in claim 1 or 2; preferably, the polynucleotide has the sequence shown in SEQ ID NO:

4.

4. A recombinant expression vector, characterized in that, It contains the polynucleotide as described in claim 3; preferably, the recombinant expression vector is a plasmid, and the backbone of the plasmid is, for example, pET28a(+).

5. A transformant, characterized in that, It comprises the polynucleotide as described in claim 3 or the recombinant expression vector as described in claim 4; preferably, the transformant is a cell, such as a prokaryotic cell, the prokaryotic cell being, for example, an Escherichia coli cell, the Escherichia coli being more preferably Escherichia coli BL21(DE3).

6. A method for preparing the carbonyl reductase as described in claim 1 or 2, characterized in that, It includes the following steps: culturing the transformant as described in claim 5 under conditions suitable for expression; Preferably, the method satisfies one or more of the following conditions: (1) The culture medium comprises the following components: 5–17 g / L yeast extract, 10–22 g / L tryptone, 3–13 g / L NaCl, and water. Preferably, the culture medium comprises the following components: 5–17 g / L yeast extract, 10–22 g / L tryptone, 3–13 g / L NaCl, 5–15 g / L glycerol, 0.5–4 g / L K₂HPO₄·3H₂O, 0.1–2 g / L MgSO₄·7H₂O, and water. More preferably, the water is… More preferably, the culture medium is LB liquid medium and / or fermentation medium. For example, the fermentation medium is formulated with 12 g / L yeast extract, 12 g / L tryptone, 3 g / L NaCl, 10 g / L glycerol, 2 g / L K2HPO4·3H2O, 0.5 g / L MgSO4·7H2O and deionized water. For example, the LB liquid medium is formulated with 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl and deionized water. (2) The culture medium also contains antibiotics, such as kanamycin, preferably in a concentration of 50 μg / mL; (3) The culture temperature is 25-37℃, for example 37℃; (4) The pH of the culture is 7.

7. The use of a carbonyl reductase in the preparation of a compound as shown in Formula I, further characterized in that the carbonyl reductase is: (1) the carbonyl reductase as described in claim 1 or 2; And / or, (2) a carbonyl reductase having the amino acid sequence shown in SEQ ID NO:1; A carbon atom marked with an asterisk (*) indicates that, when it is a chiral carbon atom, it is in the S configuration, R configuration, or a mixture thereof. R 1 For H, C6-C 10 aryl or aryl with one or more R 1-1 Replacement C6-C 10 Aryl; R 1-1 It is cyano or Where R 1-2 It is a C1-C6 alkyl group; R 2 It is protected by H or an amino group; Preferably, the compound shown in Formula I is 8. The application as described in claim 7, characterized in that, It satisfies one or more of the following conditions: (1)R 1 In the context, the C6-C 10 aryl and one or more R 1-1 Replacement C6-C 10 C6-C in aryl 10 Each aryl group can be independently phenyl or naphthyl, for example, phenyl; (2)R 1-2 In this context, the C1-C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, such as methyl; (3) The amino protecting group is Boc or Cbz; (4) The carbon atom marked with "*" is an S-configuration or R-configuration carbon atom.

9. The application as described in claim 7, characterized in that, It satisfies one or more of the following conditions: (1)R 1 For being an R 1-1 Replacement C6-C 10 Aryl, for example Preferred (2)R 1-1 for (3)R 2 It is an amino protecting group, preferably Cbz.

10. A method for preparing a compound as shown in Formula I, characterized in that, It includes the following steps: in a liquid reaction system, in the presence of a coenzyme and a carbonyl reductase, the compound shown in Formula II undergoes a reduction reaction as shown in the following formula to obtain the compound of Formula I; Among them, the carbonyl reductase, the carbon atom marked with "*", and R 1 and R 2 Each independently as described in any one of claims 7-9; Preferably, the preparation method satisfies one or more of the following conditions: (1) The liquid reaction system includes a buffer solution, preferably a phosphate buffer solution, such as a 0.1M phosphate buffer solution; (2) The liquid reaction system further includes a co-solvent, which is selected from one or more of dimethyl sulfoxide, isopropanol and toluene, such as dimethyl sulfoxide; (3) The liquid reaction system further includes a hydrogen donor, such as glucose; (4) The coenzyme is a reducing coenzyme, such as NADH and / or NADPH; or, the coenzyme is an oxidizing coenzyme and a dehydrogenase, wherein the oxidizing coenzyme may be NAD+ and / or NADP+, and the dehydrogenase may be glucose dehydrogenase. Preferably, the reducing coenzyme is obtained by the following coenzyme regeneration step: reducing NADP+ in the presence of a dehydrogenase and a hydrogen donor. + and / or NAD + ; (5) The compound shown in Formula II is More preferably, the preparation method satisfies one or more of the following conditions: (1) The volume-to-mass ratio of the buffer solution to the compound shown in Formula II is 5 to 100 mL / g, for example 37.5 mL / g; (2) The volume-to-mass ratio of the cosolvent to the compound shown in Formula II is 1 to 20 mL / g, for example 12.5 mL / g; (3) The coenzyme contains NAD+ and / or NADP+, and the mass ratio of NADP+ to the compound shown in Formula II can be (0.001 to 0.1):1, preferably (0.01 to 0.05):1, for example 0.02:1; (4) The coenzyme includes glucose dehydrogenase. When the amount of glucose dehydrogenase is calculated based on the mass of the wet bacterial cells that produce the dehydrogenase, the mass ratio of glucose dehydrogenase to the compound shown in Formula II can be (1-5):1, preferably (2-3):1, for example 2.5:

1. (5) The molar ratio of the hydrogen donor to the compound shown in Formula II is (1-5):1, preferably 2:1; (6) The carbonyl reductase is added in the form of a free enzyme, an immobilized enzyme, a bacterial powder or a bacterial cell, for example, a bacterial cell enzyme, preferably added to the transformant as described in claim 5, and more preferably, the mass ratio of the bacterial cell enzyme to the mass of the compound as shown in Formula II is (1-10):1, for example (4-6):1, and more preferably 5:1; (7) The reaction temperature of the reduction reaction is 10℃-50℃, for example 25℃ or 30℃; (8) After the reduction reaction is completed, the following post-processing steps are also included: adding a haloalkane solvent to the liquid reaction system, extracting and separating to obtain the compound shown in Formula I. The haloalkane solvent may be dichloromethane. The separation may include concentration, ester solvent extraction, washing, drying, filtration and concentration steps to obtain the compound shown in Formula I. (9) The reduction reaction preferably includes the following steps: the compound shown in Formula II, the cosolvent, the buffer solution, NADP+, glucose, the glucose dehydrogenase and the carbonyl reductase mixture are subjected to a reduction reaction at 10℃-50℃. After the reaction is completed, a haloalkane solvent is added, and the mixture is extracted and separated to obtain the compound shown in Formula I. (10) The glucose dehydrogenase is derived from the glucose dehydrogenase of Bacillus megaterium, and the glucose dehydrogenase preferably has the amino acid sequence shown in SEQ ID NO:5, and the polynucleotide encoding the glucose dehydrogenase preferably has the sequence shown in SEQ ID NO:6.

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